Proteins form the major component of insect cuticle to which they impart structural rigidity. The cuticle is an effective barrier to most microbes but entomopathogenic fungi can breach it using extracellular proteases. Consequently, fungal pathogens are the only biological means of controlling aphids and other sap-sucking insects which would not naturally ingest viruses or bacteria; and they are important for coleopteran control since viral and bacterial diseases are unknown for most beetle pests.
"World Picture of Biological Control of Insects by Fungi", Mem. Inst. Oswaldo Cruz, Rio de Janeiro, Vol. 84, Supl. III, 89-100, 1989, by Donald W. Roberts, joint inventor, is an overview of the role of entomopathegenic fungi and is incorporated herein by reference. Virtually all insect orders are susceptible to fungal diseases. Fungi are particularly important in the control of pests which feed by sucking plant juices, because these insects have no means of ingesting pathogens. There are approximately 700 species of entomopathogenic fungi in almost 100 genera (see table 1 of Roberts, 1989).
Entomopathogenic fungi frequently decimate insect populations in spectacular epizootics and are already used for the biological control of some pest species, as discussed in "Use of pathogens in insect control" by D. W. Roberts et al, In: Handbook of Pest Management in Agriculture, 2nd ed., Vol. 2 (Pimentel, D., ed.), CRC Press, Boca Raton, Fla. pp. 243-278, 1991, incorporated herein by reference. Nevertheless, the considerable potential of entomopathogenic fungi for pest control has never been realized, largely because so little is known about the genetic and molecular basis of fungal pathogenesis in insects, as discussed in "Prospects for development of molecular technology for development of molecular technology for fungal insect pathogens", by Yoder et al, In: Biotechnology in Invertebrate Pathology and Cell Culture. Academic Press, NY. pp. 197-218, 1987; and in "The potential impact of fungal genetics and molecular biology on biological control, with particular reference to entomopathogens" by J. B. Heale, In: Fungi in Biological Control Systems (M. Burge, ed.) Manchester University Press, Manchester pp. 211-234, 1988; both incorporated herein by reference. Such knowledge is urgently needed to allow the genetic engineering of fungi for use as biological control agents.
Current widely-publicized problems with synthetic chemical insecticides have given rise to a sense of urgency in the development of biological control agents as supplements or alternatives to these chemicals. Although widely used in some less-developed nations, entomopathogenic fungi have only recently assumed importance in United States agriculture and household entomology. This is in spite of the fact that entomopathogenic fungi are key regulatory factors in pest insect populations, are considered very promising biological control agents and provide the only practical means of biological control of insects which feed by sucking plant or animal juices, and for the many coleopteran pests which have no known viral or bacterial diseases. The imperfect fungus, Metarhizium anisopliae, was registered by the U.S. EPA in 1993 for cockroach control, and registration packages for use of Beauveria bassiana, another imperfect fungus, in agriculture have been submitted. Verticillium lecanii is registered in Europe, and there is interest in it in the USA, particularly for whitefly control. Any consideration of the suitability of a microorganism for commercial purposes inevitably leads to the possibility of improving its performance. This is particularly true for traditional biopesticides (viruses, bacteria, and fungi) as their performance is commonly perceived to be poor compared with chemical pesticides. However, registration requests to date have been for naturally-occurring fungi obtained by standard selection procedures and improved as pathogenic agents by developing the technologies required for optimizing production and stability of the inoculum. Improvements have also been attempted through parasexual crossing and protoplast fusion (Heale et al, 1989).
Genetic engineering using transformation and gene cloning for pathogenicity traits that are regulated by single genes or gene clusters provides the most targeted and flexible approach to dissect, and eventually alter, the physiology of entomopathogenic fungi without the cotransfer of possibly undesirable linked characteristics. The use of genetically engineered microorganisms can also produce combinations of traits that are not readily identified in nature, even if they exist somewhere. Of greatest importance, genetic engineering allows transfer of pathogenicity genes between species, genera, and even kingdoms. Entomopathogenic fungi represent an unconsidered, and therefore untapped, reservoir of pesticidal genes for the production of advanced engineered pesticides; an important consideration, given that the lack of "useful" pesticidal genes for transfer has been a major constraint in the implementation of biotechnology in crop protection (Gatehouse et al., 1992).
One of the most promising candidates for widespread commercial use is the deuteromycete, Metarhizium anisopliae ("Entomogenous Fungi" by McCoy et al, 1988 In: CRC Handbook of Natural Pesticides, Vol. 5: Microbial Insecticides Part A, Entomogenous Protozoa and Fungi (Ignoffo, C. M., ed.) CRC Press, Boca Raton, Fla. p. 151, incorporated herein by reference.). The process by which this pathogen colonizes an insect host has been examined in order to develop, through genetic manipulation, fungi which are appropriate for the control of specific insect pests. The chymoelastase proteases produced by M. anisopliae and other pathogenic fungi currently provide the best understood model of a fungal determinant of entomopathogenicity ("The role of cuticle-degrading enzymes in fungal pathogenesis in insects" by Charnley, A. K. and St. Leger, R. J, 1991, In: The Fungal Spore and Disease Initiation in Plants and Animals (Cole, G. T. and Hoch, H. C., eds.). Plenum Press, NY. pp. 267-286, incorporated herein by reference.).
The term "chymoelastase" is used hereinabove to describe an elastolytic enzyme with a primary specificity for amino acids with large hydrophobic side groups. An elastolytic enzyme, or elastase, is a functional term describing an enzyme capable of solubilizing elastin, usually with a primary specificity for amino acids with small hydrophobic side chains, e.g. alanine. By contrast, a chymoelastase with elastolytic activity has optimum activity against typical substrates for chymotrypsins, e.g. phenylalanine. Chymoelastase is a good general purpose stain remover because of its broad range of specificity for protein substrates. Pr1 has similar substrate specificity to subtilisin-like enzymes and subtilisin-like enzymes is a broad category of enzymes recognized by those skilled in the art. The primary chymoelastase produced by M. anisopliae believed to be primarily responsible for the invasion through insect cuticle has been named Pr1 and the terms "chymoelastase" and "Pr1" are used interchangeably hereinabove.
The importance of Pr1 during Metarhizium infection processes was suggested, firstly, by its considerable ability to degrade cuticle, which is attributed (at least in part) to the structural importance and assessibility of cuticular proteins ("Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae" by R. J. St. Leger, Arch. Biochem. Biophys. 253, 221-232, 1987a, incorporated herein by reference.); and, secondly, by its high level at the site of penetration before and coincident with hydrolysis of cuticle proteins ("Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca sexta) cuticle", by M. S. Goettel et al, J. Gen. Microbiol. 135, 2233-2239, 1989; "Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomituria and Manduca sexta", by R. J. St. Leger et al, J. Gen. Microbiol. 133, 1371-1382, 1987b; and "Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungus Metarhizium anisopliae" by R. J. St. Leger et al, Exp. Mycol. 13, 253-262, 1989a; all incorporated herein by reference. It has also been demonstrated that antisera against Metarhizium Pr1 or specific inhibitors of Pr1 block penetration of host cuticles and reduce infection indicating that the regulation of expression of the Pr1 gene may determine the capacity of the fungus to cause disease ("Role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta" by R. J. St. Leger et al, J. Invertebr. Pathol. 52, 285-293, 1988a, incorporated herein by reference).
The purified Pr1 has been characterized for substrate specificity and inhibition by typical serine protease inhibitors (St. Leger et al., 1987b). Utilization of pathogen enzymes, particularly Pr1, has assisted investigators to understand how the cuticle is degraded naturally, and it is predicted that further characterization will enable manipulation of enzyme levels using chemical and biotechnological procedures for the purpose of insect control ("Insect cuticle structure and metabolism", by Kramer et al., In: Biotechnology for Crop Protection (Hedin, P. A., Menn, J. J. and Hollingworth, R. M., eds.) American Chemical Society, Washington pp. 160-183, 1988, incorporated by reference). For example, the characterization of genes for anticuticular enzymes is an important step towards engineering vectors for introduction of these genes into other microbes and plants.
Potentially, specialization for a pathogenic lifestyle may operate by way of regulatory controls which allow expression of genes under conditions in which similar genes are not expressed in non-pathogens. Previously, it was demonstrated that antisera against Metarhizium Pr1 or specific inhibitors of Pr1 block penetration of host cuticles and reduce infection indicating that the level of active Pr1 may determine the capacity of the fungus to cause disease (St. Leger et al., 1988a). Production of the enzyme without differentiation of infection structures can occur rapidly by nutrient deprivation alone ("Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae", by R. J. St. Leger et al, Archives Microbiology 150: 413-416, 1988b, incorporated herein by reference.) making Metarhizium an amenable system for the study of the molecular controls of protein synthesis.